Cell-free protein synthesis system from Escherichia coli cells cultured at Decreased temperatures improves productivity by decreasing DNA template degradation



Cell-free protein synthesis system from Escherichia coli cells cultured at Decreased temperatures improves productivity by decreasing DNA template degradation

Introduction

Cell-free protein synthesis is a powerful protein production method with several advantages. We have been improving the cell-free system using an Escherichia coli (E. coli) cell extract. We have succeeded in producing high levels of protein, about 7 mg of protein from a 1 ml reaction mixture, and synthesizing a variety of proteins. Presently, the cell-free system is one of the major protein production methods utilized by our group and for various applications such as the production of metal-binding proteins, stable-isotope labeled proteins for NMR analysis, and seleno-methionine substituted proteins for X-ray crystallography. In combination with automation technology, we have established a high-throughput protein production pipeline for structural genomics research, based on the cell-free system. (Zimmer, 2002)

Background

Various factors are present within the E. coli cell extract. Despite their negative effects on protein synthesis, some of these factors are essential for cell extract preparation, because they are required during the cell growth and/or extract preparation stage. For instance, degradation factors for DNA (deoxyribonuclease; DNase), mRNA (ribonuclease; RNase), and proteins (protease), or depression/inhibition factors for transcription and/or translation are included among these negative factors. In the case of stable isotope labeling, factors involved in amino acid metabolism are known to cause the scrambling of the label in the wheat germ cell-free system.

Several approaches have been developed to overcome these negative factors. One of the approaches is the preparation of cell extracts using modified E. coli strains in which the gene encoding the negative factor on the chromosomal DNA is partially or fully deleted , , , and . Although this approach improves the productivity of the cell-free protein synthesis, these mutant strains generally exhibit defects in translational activity or cell growth. Multiple deletions or mutations of factors can sometimes cause severe defects in cell growth. In addition, the removal of some factors can have an unfavorable effect on the cell extract preparation. For example, the cell extract prepared from a rib-deleted strain exhibited low protein production activity with a high background, due to the residual endogenous DNA. Another approach is improving the resistance of the linear DNA or mRNA template to degradation, and. These approaches, however, may limit the sequence or the conditions of the reaction, and thus narrow the applicability of the cell-free system. (Zimmer, 2002)

Main Idea

The productivity of cell-free protein synthesis using linear DNA templates is generally lower than that from plasmid DNA templates, especially when using an Escherichia coli cell extract. In the present study, we found that a simple modification of the protocol for cell extract preparation from E. coli, just by altering the cultivation temperature (37 C) of the cells to a moderately lower range (20-34 C), dramatically reduced the linear DNA degradation activity in the cell extract. This modification greatly improved the productivity of cell-free protein synthesis from linear DNA ...